1. Field of the Invention
The present invention relates to the preparation of novel glycosides of tumor-associated carbohydrate haptens.
2. Description of the Background Art
Tumor Associated Carbohydrate Antigenic Determinants. Numerous antigens of clinical significance bear carbohydrate determinants. One group of such antigens comprises the tumor-associated mucins (Roussel, et al., Biochimie 70, 1471, 1988).
Generally, mucins are glycoproteins found in saliva, gastric juices, etc., that form viscous solutions and act as lubricants or protectants on external and internal surfaces of the body. Mucins are typically of high molecular weight (often&gt;1,000,000 Dalton) and extensively glycosylated. The glycan chains of mucins are O-linked (to serine or threonine residues) and may amount to more than 80% of the molecular mass of the glycoprotein. Mucins are produced by ductal epithelial cells and by tumors of the same origin, and may be secreted, or cell-bound as integral membrane proteins (Burchell, et al., Cancer Res., 47, 5476, 1987; Jerome, et al., Cancer Res., 51, 2908, 1991).
Cancerous tissues produce aberrant mucins which are known to be relatively less glycosylated than their normal counter parts (Hull, et al., Cancer Commun., 1, 261, 1989). Due to functional alterations of the protein glycosylation machinery in cancer cells, tumor-associated mucins typically contain short, incomplete glycans. Thus, while the normal mucin associated with human milk fat globules consists primarily of the tetrasaccharide glycan, gal .beta.1-4 glcNAcp1-6(gal .beta.1-3) gal NAc-.alpha. and its sialylated analogs (Hull, et al.), the tumor-associated Tn hapten consists only of the monosaccharide residue, .alpha.-2-acetamido-3-deoxy-D-galactopyranosyl, and the T-hapten of the disaccharide .beta.-D-galactopyranosyl-(1-3).alpha.-acetamido-2-deoxy-D-galactopyranosy l. Other haptens of tumor-associated mucins, such as the sialyl-Tn and the sialyl-(2-6)T haptens, arise from the attachment of terminal sialyl residues to the short Tn and T glycans (Hanisch, et al., Biol. Chem. Hoppe-Seyler, 370, 21, 1989; Hakormori, Adv. Cancer Res., 52:257, 1989; Torben, et al., Int. J. Cancer, 45 666, 1980; Samuel, et al., Cancer Res., 50, 4801, 1990).
The T and Tn antigens (Springer, Science, 224, 1198, 1984) are found in immunoreactive form on the external surface membranes of most primary carcinoma cells and their metastases (&gt;90% of all human carcinomas). As cancer markers, T and Tn permit early immunohistochemical detection and prognostication of the invasiveness of some carcinomas (Springer). Recently, the presence of the sialyl-Tn hapten on tumor tissue has been identified as an unfavorable prognostic parameter (Itzkowitz, et al. Cancer, 66, 1960, 1990; Yonezawa, et al., Am. J. Clin. Pathol., 98 167, 1992). Three different types of tumor-associated carbohydrate antigens are highly expressed in common human cancers. The T and Tn haptens are included in the lacto series type, and type 2 chains. Additionally, cancer-associated ganglio chains and glycosphingolipids are expressed on a variety of human cancers.
The altered glycan determinants displayed by the cancer associated mucins are recognized as non-self or foreign by the patient's immune system (Springer). Indeed, in most patients, a strong autoimmune response to the T hapten is observed. These responses can readily be measured, and they permit the detection of carcinomas with greater sensitivity and specificity, earlier than has previously been possible. Finally, the extent of expression of T and Tn often correlates with the degree of differentiation of carcinomas. (Springer).
Carbohydrate-Protein Conjugates. Because the tumor-associated antigens are useful in diagnosis and monitoring of many types of carcinomas, and may also be useful in treatment, many workers have synthesized glycosides of the carbohydrate haptens and of their sialylated analogs and have used these glycosides to conjugate the haptens to proteins or synthetic peptide carriers. The glycosides have generally included an aglycon moiety from which a highly reactive functionality can be generated without altering the saccharide portion of the respective hapten glycoside. The "activated" hapten glycosides are then reacted with amino groups of the proteins or synthetic peptide carriers to form amide of Schiff base linkages. The Schiff base grouping can be stabilized by reduction with a borohydride to form secondary amine linkages; the whole coupling process is then referred to as reductive amination. (Gray, Arch. Biochem. Biophys., 163, 426, 1974).
Lemieux et al. disclosed artificial antigens in which a T-antigenic determinant is coupled to a protein or polysaccharide carrier by means of an .alpha.-O-glycosidically linked --O--(CH.sub.2).sub.n COR linking arm (U.S. Pat. Nos. 4,794,176; 4,866,045; 4,308,376; 4,362,720; 4,195,174; Can. J. Chem., 57, 1244, 1979). In this process, a D-galactal derivative is converted by azidonitration into a 2-azido-2-deoxy-D-galactopyransoyl nitrate which reacts with quaternary ammonium halides to form a 2-azido-2-deoxy-D-galactopoyranosyl halide. This halide is used as a glycosyl donor to form an .alpha.-glycoside with the alcohol, 9-hydroxynonanoic acid ethyl (or methyl) ester (Lemieux, et al., U.S. Pat. No. 4,137,401). In subsequent steps, the 2-azido-2-deoxy-D-galactopyranosyl unit is converted into the 2-acetamido-deoxy-D-galactopyranosyl unit. This can be suitably protected to attach additional glycosyl residues, such as the .beta.-D-galactopyranosyl residue at 0-3 to form the T-hapten. Alternatively, the 2-acetamido-2-deoxy-.alpha.-D-galactopyranosyl glycoside may also be used directly as the Tn hapten.
To "activate" the linker arm, the 9-glycosyloxy fatty acid ester is converted into a 9-glycosyloxy fatty acid hydrazide. The hydrazide is oxidized to the 9-glycosyloxy-nonanoic acid azide which reacts, much like an acid halide, with amino groups of proteins or synthetic peptide carriers, to bind the hapten glycoside in an amide linkage.
The Lemieux process requires a 2-azido-2-deoxygalactosyl intermediate to enable the formation of the desired .alpha.-glycoside. Also, the ester group on the linking arm is frequently unstable during chemical manipulation required for the attachment of additional glycosyl residues to the 2-acetamido-2-deoxy-D-galactopyranosyl glycoside. Due to the multi-step nature of the process, over-all yields are low, and particularly the final coupling step of acyl azide to the protein or synthetic peptide carried can be inefficient, resulting in wastage of these extremely expensive hapten glycosides.
The 2-azido-2-deoxy-D-galactopyranosyl halide intermediate required for the preparation of the T and Tn haptens according to the process of Lemieux may be directly prepared by azidochlorination of a D-galactal derivative (Naicker, et al., U.S. Pat. No. 4,935,503). Another route to 2-azido-2-deoxy-D-galactopyranosyl halides has been described by Paulsen, et al., Chem. Ber., 111, 2358, 1978). The reaction of 1,6;2,3-dianhydro-D-talopyranose (James, J. Chem. Soc., 625, 1946) with sodium azide affords a derivative of 2-azido-2-deoxy-D-galacto-pyranose which may be further converted into a glycosyl halide donor suitable for glycosylation of the Lemieux linker arm 9-hydroxynonanoic acid methyl (or ethyl) ester or an equivalent linker moiety.
Several other linking arms for conjugating haptens to proteins or synthetic peptide carriers are known to the art (Kolar, U.S. Pat. No. 4,442,284, amino acid; Feizi, U.S. Pat. No. 4,563,445, alkyl, hydroxyl alkyl, alkenyl or ether linker; Koganty, U.S. Pat. No. 5,055,562, a covalent linker comprising at least one fluorocarbon chain).
Jennings et al., U.S. Pat. No. 4,356,170, derive their carbohydrate haptens from naturally-occurring bacterial polysaccharides. Activation of the hapten is effected by controlled periodate oxidation of vicinal hydroxyl groups to form aldehyde functions. The reductive amination procedure is used to conjugate the haptens to the proteins or synthetic peptide carriers. The process of Jennings, et al., is unsuitable for preparing conjugates of the T and Tn haptens because the haptens are not readily available in pure form from natural sources, and periodate oxidation would presumably destroy the T and Tn epitopes.
Roy, et al., in U.S. Pat. No. 5,034,516, have disclosed conjugates containing carbohydrate haptens, prepared by synthesis of allyl glycosides which were subsequently co-polymerized with suitable co-monomers such as acrylamide (Kochetkov, Pure and Appl. Chem., 56, 923, 1984). However, the resulting co-polymeric conjugates are often poorly immunogenic, and the method of Roy, et al., does not permit the attachment of the haptens to the desired protein or synthetic peptide carriers.
Bernstein, et al. (Carbohydr. Res., 78, C1-C3, 1980) disclosed ozonolytic cleavage of allyl glycosides of carbohydrate haptens to produce aldehyde glycoside derivatives which may be coupled to proteins or peptide carriers by reductive amination. However, ozonolytic cleavage of allyl glycosides results in the formation of formaldehyde as a by-product of the desired hapten glycoside aldehyde derivatives. Formaldehyde contributes to denaturation of the protein carriers and competes with the hapten glycoside aldehyde derivatives for available amino groups of the proteins or peptide carriers. Unfortunately, because formaldehyde is strongly hydrated and water soluble, there is no simple means of removing formaldehyde from the solutions containing the hapten glycoside aldehyde derivatives.
Several groups of investigators have reported methods for preparation of the sialyl (2-6)T and sialyl-Tn antigens. Paulsen, et al., Carbohydr. Res., 137, 63, 1985) describe the synthesis of the disaccharide .alpha.-sialyl-(2-6)-.beta.-2-acetamide-2-deoxy-D-galactopyranose. Lijima, et al. Carbohydr. Res., 172, 183, 988) disclosed the synthesis of the sialyl-Tn hapten as a glycoside of L-serine, using as an intermediate a protected allyl glycoside of sialyl-(2-6)-2-azido-2-deoxy-D-galactopyranose.
Conjugation of sialic acid-containing oligosaccharide haptens to carriers by the Lemieux process is highly impractical due to the difficulty in distinguishing the carboxylic ester functions on sialic acid and on the linker arm.
Thus, the process taught by prior workers for preparing glycoconjugate antigens comprising the Tn, T, sialyl-Tn, and sialyl-(2.fwdarw.6)T haptens involve expensive starting materials such as D-galactal derivatives or 1,6,2,3-dianhydro-D-talose which are processed to the desired glycoconjugates in multi-step reaction sequences with low over-all yields. Use of these processes for preparing the required glycoconjugates in commercial quantities of pharmaceutical grade purity is not practical. There is therefore a need for a process that provides these important glycoconjugates in relatively large quantities and at reasonable cost.
Glycosoylation Methods
The chemical synthesis of oligosaccharide, especially in a stereochemically controlled manner, can be challenging. The classical Koenigs-Knorr method, developed in 1901, involves glycosylating a sugar (the glycosyl acceptor) with a glycosyl bromide or chloride, using a heavy metal salt catalyst. A large number of alternatives are discussed by Schmidt, Angew. Chem. Int. Ed. Engl. 25:212-35 (1988) who in passing discusses Fischer-type glycosylations, which are acid catalyzed. He comments that this method "does not involve an isolable intermediate and, partly as a result of its reversibility has attained hardly any significance for the synthesis of complex saccharides". Thus, Schmidt considers and rejects the Fischer approach.
Schmidt observes that 2-amino sugars, especially N-acetylglucosamine and N-acetylgalactosamine, are of great importance in biologically occurring complex oligosaccharides and glycoconjugates. He advocates glycosidic coupling of GlcNAc via the trichloroacetimidate method, with various catalysts. A person of ordinary skill in the art would reasonably infer that this is Schmidt's preferred approach to GalNAc coupling, too.
Flowers, Meth. Enzymol., 138:359 (1987) also alludes (pp. 373-3) to the Fischer-type glycosylation. While he indicates that "the preferred stability of .alpha.-D-glycopyranosides in most cases enables their isolation in reasonable yield," he cautions that "this approach is usually not feasible for glycosides of disaccharides, since alcoholysis of the interglycosidic linkage competes with glycosylation of the reducing OH." He concludes that "complex mixtures often result" from Fischer-type glycosylation.